Application of P450 monooxygenase in increasing content of fucoxanthin in microalgae

Abstract

The invention belongs to the technical field of biology, and discloses application of P450 monooxygenase in increasing the content of fucoxanthin in microalgae, the microalgae with the fucoxanthin synthesis and / or light stress response capacity improved and a construction method of the microalgae. Researches find that P450 monooxygenase is closely related to synthesis of fucoxanthin and plays a regulating role in light stress response of diatom, and overexpression of P450 monooxygenase can cause increase of the content of fucoxanthin in microalgae; under the condition of light stress, the content of fucoxanthin in a transformant is greatly increased compared with that of wild type fucoxanthin. According to the invention, an engineering algal strain with improved fucoxanthin synthesis and / or light stress response capability is also obtained, a material basis is provided for the biosynthesis industrialization of fucoxanthin, and the additional value of microalgae culture is improved at the same time.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to the application of P450 monooxygenase in increasing the content of fucoxanthin in microalgae. Background Art

[0002] Fucoxanthin, also known as fucoxanthin, is the second largest carotenoid in nature. In recent years, it has been favored in the development of marine food nutritional factors because of its significant effects in anti-oxidation, antibacterial, anti-cancer, anti-inflammatory, hypoglycemic, and obesity inhibition. In addition, fucoxanthin has important applications in the capture of light energy in photosynthesis. Blue-green light has strong penetration ability in seawater, so most marine algae contain fucoxanthin, which is used to capture visible light in the blue-green band. If fucoxanthin is transferred to other photosynthetic organisms, it can be used to broaden the light capture spectrum and improve the efficiency of photosynthesis.

[0003] Fucoxanthin is a substance unique to algae, and no artificial substitute has been found so far. Fucoxanthin has been used in commercial weight management products abroad, such as those from Solaray® and fucoTHIN®, but these products mainly use fucoxanthin derived from brown algae. Brown algae as a source of fucoxanthin is not only expensive and seasonally restricted, but also has a very low content (<0.1% DW). Therefore, the development of other groups of microalgae as alternative sources for fucoxanthin production has become a hot topic. Among the many algae groups, diatoms are expected to become a source of sustainable production of fucoxanthin and an ideal cell factory due to their high fucoxanthin content (about 1%-6%), which is more than 100 times that of brown algae, as well as their high productivity and good quality. However, in diatom cells, the synthesis pathway of fucoxanthin is currently unclear, and some key intermediate pigments involved in the pathway and related pigment synthase genes have not been fully revealed. Summary of the invention

[0004] The present invention aims to solve the problems in the prior art and provides the use of P450 monooxygenase in increasing the content of fucoxanthin in microalgae. The purpose of the present invention is to provide a gene with high activity and catalysis of fucoxanthin synthesis, and to increase the synthesis of fucoxanthin in microalgae through bioengineering methods.

[0005] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides the use of P450 monooxygenase in increasing the content of fucoxanthin in microalgae, wherein the P450 monooxygenase is the protein in (i) or (ii) or (iii): (i) a protein synthesized by the amino acid sequence shown in SEQ ID NO.1; (ii) a derivative protein synthesized from the amino acid sequence shown in SEQ ID NO.1 by substitution and / or deletion and / or addition of one, several or dozens of amino acid residues and having the same function as (i); (iii) a derivative protein having more than 90% homology with the amino acid sequence shown in SEQ ID NO.1 and having the same function as (i).

[0006] The present invention discloses a new function of P450 monooxygenase derived from P. tricornutum CCMP2561. P450 monooxygenase or the corresponding Pt4417 gene has the function of increasing the content of fucoxanthin in microalgae, and can be used to increase the content of fucoxanthin in microalgae. The substantial increase in the content of fucoxanthin will also be beneficial to downstream extraction, purification and other process flows.

[0007] The inventors knocked down and overexpressed P450 monooxygenase in microalgae, compared the fucoxanthin production of the knockdown, overexpression and wild-type P. triangularis, and found that the fucoxanthin content in the knockdown P. triangularis was lower than that in the wild type, while the fucoxanthin content in the two overexpression P. triangularis was increased by 21.24% and 22.62% respectively compared with the wild type. It can be seen that the expression level of P450 monooxygenase will cause the decrease and increase of fucoxanthin synthesis, confirming that the Pt4417 gene or P450 monooxygenase is involved in the synthesis of fucoxanthin.

[0008] The P450 monooxygenase involved in the application claimed in the present invention may be: (i) a P450 monooxygenase consisting of 584 amino acid residues, whose amino acid sequence is shown in SEQ ID No.1.

[0009] Amino acid sequence SEQ ID No.1: .

[0010] The P450 monooxygenase involved in the application claimed in the present invention can also be a protein derived from (i) which is obtained by replacing and / or deleting and / or adding one or more (such as 1-30; preferably 1-20; more preferably 1-10, such as 5, 3) amino acid residues of the amino acid sequence of SEQ ID NO.1, and has the same function as (i) protein; or a protein derived from (i) which has 90% (preferably more than 90%, such as 95%, 98%, 99% or higher) homology with the protein sequence defined in (i) and has the function of (i) protein.

[0011] Preferably, the P450 monooxygenase increases the fucoxanthin content in microalgae under light stress conditions.

[0012] The P450 monooxygenase involved in the present invention can improve the light stress response ability of microalgae. Under light stress conditions, the fucoxanthin content in the overexpressed triangular phytosalis is increased by 99.21%-106.28% compared with the wild type.

[0013] Preferably, the gene sequence of the P450 monooxygenase is shown as SEQ ID NO.2.

[0014] The gene whose sequence is shown in SEQ ID NO.2 is a Pt4417 gene, which consists of 1855 bases and has a nucleotide sequence of:

[0015] The present invention protects the function of P450 monooxygenase. The coding gene of the P450 monooxygenase can be the above-mentioned Pt4417 gene, or a homologous gene with high homology to SEQ ID NO.2 (such as homology higher than 90%; preferably higher than 95%; more preferably higher than 98%).

[0016] The microalgae include, but are not limited to, diatoms, brown algae and green algae.

[0017] Preferably, the microalgae are diatoms.

[0018] More preferably, the diatom is Phaeodactylum triangularis ( Phaeodactylum tricornutum ).

[0019] In a second aspect, the present invention also provides a method for improving the ability of microalgae to produce fucoxanthin, which comprises overexpressing a P450 monooxygenase having the amino acid sequence described in (i) or (ii) or (iii) below in the microalgae: (i) a protein synthesized by the amino acid sequence shown in SEQ ID No. 1 in the sequence listing; (ii) the amino acid sequence shown in SEQ ID No. 1 in the sequence listing is substituted and / or deleted and / or added with one, several or dozens of amino acid residues, and the derived protein has the same function as (i); (iii) a derivative protein having more than 90% homology with the amino acid sequence shown in SEQ ID NO.1 and having the same function as (i).

[0020] Preferably, the gene of the P450 monooxygenase with the amino acid sequence as shown in SEQ ID NO. 1 is introduced into the microalgae by genetic engineering method, and the gene is overexpressed in the microalgae.

[0021] More preferably, the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

[0022] In a third aspect, the present invention also provides a method for constructing microalgae with improved fucoxanthin synthesis and / or light stress response ability, comprising the step of overexpressing a P450 monooxygenase having an amino acid sequence as shown in SEQ ID NO.1 in the microalgae.

[0023] Preferably, the nucleotide sequence of the P450 monooxygenase gene is as shown in SEQ ID NO.2; the nucleotide sequence shown in SEQ ID NO.2 is connected to an overexpression vector and then transformed into microalgae.

[0024] The microalgae include, but are not limited to, diatoms, brown algae and green algae.

[0025] More preferably, the microalgae is Phaeodactylum tricornutum; the overexpression vector is a microalgae expression vector or a plant expression vector, and a constitutive or inducible promoter drives the expression of the P450 monooxygenase gene in the microalgae.

[0026] Specifically, the constitutive or inducible promoter is selected from one of the following promoters: fucoxanthin-chlorophyll a / c binding protein promoter, endogenous β-tubulin promoter, β-actin promoter, ribulose bisphosphate carboxylase-oxygenase promoter and nitrate reductase promoter.

[0027] In a fourth aspect, the present invention further provides a microalgae with improved fucoxanthin synthesis and / or light stress response ability, which is constructed by the above method.

[0028] The engineered microalgae containing P450 monooxygenase genes all fall within the protection scope of the present invention.

[0029] In a fifth aspect, the present invention also provides the use of the above-mentioned microalgae in the production of fucoxanthin.

[0030] Fucoxanthin can be produced by enriching and culturing the microalgae provided by the present invention, obtaining the algae, and then crushing and extracting the fucoxanthin. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] Figure 1 The quantitative analysis results of fucoxanthin in the triangular flavonoids overexpressing Pt4417 in Example 2; Figure 2 The quantitative analysis results of fucoxanthin in the Pt4417 knocked down Phaeodactylum triangularis in Comparative Example 1; Figure 3 The results of quantitative analysis of fucoxanthin in the P. triangularis overexpressing Pt4417 cultured under high light in Example 3. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] The sources of Phaeodactylum triangularis used in the following embodiments of the present invention are: Phaeophyta triangularis Phaeodactylum tricornutum )CCMP2561 was provided by the Bigelow National Center for Marine Algae and Microbiota.

[0035] Cultivation method: The triangular brown algae CCMP2561 was inoculated into F / 2 culture medium at a temperature of 22°C and a light intensity of 40 μEm -2 s -1 The culture was carried out under the conditions of a shaking incubator with a speed of 150 rpm.

[0036] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can be obtained through commercial channels.

[0037] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0038] Unless otherwise specified, the implementation of the present invention will use conventional botanical techniques, microorganisms, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination and bioinformatics techniques that are obvious to those skilled in the art. These techniques are fully explained in the published literature. In addition, the methods of DNA extraction, construction of phylogenetic trees, gene editing methods, construction of gene editing vectors, and obtaining gene-edited plants used in the present invention can be achieved by using methods already disclosed in existing literature, except for the methods used in the following examples.

[0039] Example 1 Acquisition of P450 monooxygenase genes The triangular algae CCMP2561 were inoculated into F / 2 culture medium at 22°C and a light intensity of 40 μE m -2 s -1 The cells were cultured at a shaking speed of 150 rpm. After the cells reached the logarithmic phase, they were harvested by centrifugation (2000 × g, 5 min) for about 10 min. 7Cells were ground and broken in the presence of liquid nitrogen, and then total RNA was extracted using the TRI Reagent kit (Invitrogen, Carlsbad, CA, USA). The concentration of total RNA was determined using NannoDrop 2000c (Thermo Scientific, Wilmington, Delaware, USA), and the quality was tested by gel electrophoresis. 1 μg of total RNA was reverse transcribed to synthesize cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen) kit, and the method was based on the steps in the kit manual. The cDNA synthesized by reverse transcription was used as a template, and PCR amplification was performed under the action of a high-fidelity DNA polymerase to obtain the full-length coding sequence of the Pt4417 gene of Pt4417. The nucleotide sequence of the Pt4417 gene is shown in the sequence SEQ ID NO.2, which consists of 1855 bases, and the corresponding amino acid sequence of the P450 monooxygenase is shown in the sequence SEQ ID NO.2, which consists of 584 amino acids.

[0040] The primers used for PCR amplification are: Forward primer: 5'-cga gaattc GCCACCATGCTCGTGAACCTTACA-3'; Reverse primer: 5'-ggtacc gagctc CAGCAACGAGAATTTTCGAACC-3′; It includes not only the sequences overlapping with the Pt4417 cDNA sequence (uppercase), but also the sequences homologous to the target vector (lowercase).

[0041] PCR system: 2×Master Mix 10 µL; template DNA 1 µL; 10 μM upstream primer 1 µL; 10 μM downstream primer 1 µL; add ddH2O to make the total system 20 µL.

[0042] PCR cycles: (1) 98°C, 30 s; (2) 98°C, 10 s; (3) 65°C, 10 s; (4) 72°C, 2 min; (5) 30 cycles; (6) 72°C, 10 min.

[0043] After the amplified DNA sequence was purified, restriction endonuclease EcoRI and SacI The fragment was digested with enzymes, purified, recovered, cloned into pUC-19 vector, and sequenced for verification.

[0044] Example 2 This embodiment provides a microalgae with improved fucoxanthin synthesis and / or light stress response ability, and a construction method thereof, comprising the following steps: (1) Construction of overexpression vector of P. triangularis Pt4417 The amplified Pt4417 sequence was digested with restriction enzymes EcoRI and SacI, then purified and recovered and cloned into the corresponding restriction sites of the overexpression vector pPha-T1-eGFP to obtain the plasmid pPha-T1-Pt4417-eGFP, which was then sequenced and verified.

[0045] (2) Transformation of pPha-T1-Pt4417-eGFP The triangular brown finger algae CCMP2561 was cultured in F / 2 liquid medium to the logarithmic phase. 25 mL of algae liquid was collected for each electroporation reaction by centrifugation (2000×g, 10 min, 4°C). After removing the supernatant, 1 mL of ice-cold 375 mM sterile Sorbitol was added to the centrifuge tube to resuspend the algae cells and transfer them to a sterilized 1.5 mL centrifuge tube, mix thoroughly, and then centrifuge (2000×g, 10 min, 4°C). The supernatant was discarded and washed twice with ice-cold 375 mM sterile Sorbitol. After washing, the supernatant was discarded, 100 μL of 375 mM Sorbitol was added to resuspend, and 5 μg of linearized plasmid pPha-T1-Pt4417-eGFP was added. The mixture was flicked to mix, placed on ice for 30 min, and then the mixture was transferred to a 2 mm electroporation cup for electroporation. The electroporation parameters were 500 V, 25 μF, and 400 Ω. After electroporation, the cells were immediately transferred to a 15 mL sterile centrifuge tube containing 10 mL F / 2 medium and incubated at 22°C and 40 μE m -2 s -1 Light intensity culture for 24 hours. Take out the 15 mL centrifuge tube containing algae liquid, centrifuge (4000×g, 10 min, 4℃), discard 9 mL supernatant, mix the remaining concentrated algae liquid evenly, take 200 μL and use a sterile glass rod to evenly spread on a resistance plate containing 100 μg / mL zeocin for screening. Generally, after 3-4 weeks, resistant clones can be seen on the plate with the naked eye. After 4-5 weeks, resistant clones can be picked in a 24-well plate, and liquid F / 2 medium containing 37.5 μg / mL zeocin can be added for culture, and the positive transformants screened will be preserved.

[0046] (3) Expression of Pt4417 gene The positive transformants were cultured in liquid F / 2 medium containing 37.5 μg / mL zeocin until the logarithmic phase and then centrifuged (2000×g, 5 min) to collect about 5×10 7cells for total RNA extraction, cDNA synthesis and subsequent real-time fluorescence quantitative PCR. The total RNA extraction and cDNA synthesis were performed according to the steps in Example 1. Real-time fluorescence quantitative PCR was performed using SYBR Green PCR Master Mix (Invitrogen), and the specific steps were performed according to the instructions. Pt4417 overexpression was normalized using the internal reference gene β-actin.

[0047] The forward primer and reverse primer used for Pt4417 are: Forward primer: 5′-TTGTCGCAGTCATCCGTCTC-3′; Reverse primer: 5′-AGGGGGACGGGATAAAGTCA-3′.

[0048] The forward primer and reverse primer used for β-actin are: Forward primer: 5′-TATTGTTCATCGCAAGTGCTTCTAA-3′; Reverse primer: 5′-TAATACACCTCCTACAAACGTTGAAGA-3′.

[0049] (4) Fucoxanthin extraction and content analysis After culturing two positive transformants for 3 days, fresh algae were collected by centrifugation at 3000×g for 5 min, and then 6 mL of chloroform: methanol (2:1; volume ratio) mixture was added. After resuspending, it was placed on a vortex shaker for 30 min and centrifuged at 5000×g for 5 min to collect the supernatant. Repeat this step until the algae is colorless. Then, add 0.75% NaCl solution of 1 / 4 of the total volume of the collected supernatant and vortex for 5 min. Subsequently, centrifuge at 4°C and 3000 rpm for 5 min, and absorb the organic phase at the lower layer to obtain the pigment extract. Finally, the extract was blown dry using a nitrogen gas meter and stored at -80°C for future use. The dried extract was dissolved in a ratio of 100 μL of acetone per 1 mg of biomass. After filtration, 200 μL was taken in a sample injection bottle for quantitative analysis.

[0050] Waters high performance liquid chromatography (HPLC) was used for detection, with a C18 column (4.6 mm×250 mm) and a 2996 photodiode array detector. Mobile phase A: ethyl acetate / methanol (32:68); phase B: acetonitrile / methanol / 0.1 M Tris (84:2:14). The elution gradient was: 0-10 min phase A increased from 0 to 100%, maintained for 15 min, 25-28 min phase A decreased to 0, maintained for 5 min. The injection volume was 10 μL, and the flow rate was 1.2 mL / min. Fucoxanthin was identified based on the absorption spectrum and retention time, and quantified using a standard curve. The results are shown in Figure 1 shown.

[0051] Figure 1 It can be seen that the fucoxanthin content of the engineered algae strains Pt4417-OE1 and Pt4417-OE2 provided in this example increased by 21.24% and 22.62% respectively compared with the wild type. From the results, it can be seen that overexpression of p450 enzymes in P. tricornutum leads to an increase in fucoxanthin content, indicating that the Pt4417 gene or p450 enzyme can promote the synthesis of fucoxanthin.

[0052] Comparative Example 1 This comparative example provides an engineered algae strain and a construction method, which comprises the following steps: The cDNA template was obtained in Example 1, and specific primers were designed according to the sequence of Pt4417, and the 200 bp fragment at the 3' end was selected as the target sequence of the interfering RNA (shRNA). The primer sequences are as follows: RiPt4417-F1:5'-atcctggccggataa catatg GAATCCGCGGGTGTCGTC-3′; RiPt4417-R1:5'-cttttgCAATACACGAGCGGTGGTATCC-3'; RiPt4417-F2:5'-ccgctcgtgtattgCAAAAGACCTTGTCCCAGCG-3'; RiPt4417-F2:5'-agtcgaggtagctca gaattc GAATCCGCGGGTGTCGTC-3'.

[0053] After the amplified DNA sequence was purified, it was digested with restriction enzymes NdeI and EcoRI, and then purified and recovered. The long and short fragments of the target gene were connected to the knockdown vector pKS-fcpB-RNAi using a multi-fragment homologous recombinase to generate a recombinant knockdown vector pKS-fcpB-RNAiPt4417, which was sequenced and verified. The transformation of pKS-fcpB-RNAiPt4417 was performed using the method in Example 2, and the gene expression and fucoxanthin content were analyzed. The results are shown in Figure 2. Figure 2 shown.

[0054] The results showed that the fucoxanthin content of the engineered algae strains Pt4417-KD1 and Pt4417-KD2 provided in this comparative example was reduced by 30.42% and 34.5% respectively compared with the wild type. Combined with the results in Example 2, it can be seen that knocking down and overexpressing P450 monooxygenase will cause a decrease and increase in fucoxanthin synthesis, confirming that the Pt4417 gene or P450 monooxygenase is related to the synthesis of fucoxanthin.

[0055] Example 3 This example provides the use of P450 monooxygenase in increasing the content of fucoxanthin in microalgae under light stress conditions.

[0056] The microalgae with improved fucoxanthin synthesis and / or light stress response ability provided in Example 2 were placed in a 300 μE m - 2 s -1 After three days of cultivation, the algae were collected by centrifugation (2000×g, 10 min, 4°C) and the fucoxanthin content was analyzed according to the method in Example 2. Figure 3 shown.

[0057] The results showed that under high light conditions, the fucoxanthin content in both wild-type and overexpression engineered algae decreased. The wild-type algae was severely affected by high light, with a 73.49% reduction in fucoxanthin content, while the fucoxanthin content in the overexpression engineered algae decreased by 54.89% and 56.93%, respectively. The fucoxanthin content in the overexpression engineered algae increased by 106.28% and 99.21% compared with the wild-type. This indicates that the Pt4417 gene or P450 monooxygenase can improve the light stress response ability of the algae and promote the synthesis of fucoxanthin under high light conditions.

[0058] In summary, P450 monooxygenase is closely related to the synthesis of fucoxanthin and can play a regulatory role in the light stress response of microalgae. The content of fucoxanthin in microalgae can be increased by overexpressing P450 monooxygenase in microalgae. The engineered algae strains with improved fucoxanthin synthesis and / or light stress response ability obtained by genetic engineering can produce high fucoxanthin and can be used in the production of fucoxanthin.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of P450 monooxygenase in increasing the content of fucoxanthin in microalgae, characterized in that: The P450 monooxygenase is the protein in (i) or (ii) or (iii): (i) a protein synthesized by the amino acid sequence shown in SEQ ID NO.1; (ii) a derivative protein synthesized from the amino acid sequence shown in SEQ ID NO.1 by substitution and / or deletion and / or addition of one, several or dozens of amino acid residues and having the same function as (i); (iii) a derivative protein having more than 90% homology with the amino acid sequence shown in SEQ ID NO.1 and having the same function as (i).

2. The use according to claim 1, characterized in that: The P450 monooxygenase increases the fucoxanthin content in microalgae under light stress conditions.

3. The use according to claim 1, characterized in that: The gene sequence of the P450 monooxygenase is shown in SEQ ID NO.2; and / or The microalgae is diatom; preferably, the diatom is Phaeodactylum triangularis ( Phaeodactylum tricornutum ).

4. A method for improving the fucoxanthin production capacity of microalgae, comprising overexpressing a P450 monooxygenase having the amino acid sequence described in (i) or (ii) or (iii) below in the microalgae: (i) a protein synthesized by the amino acid sequence shown in SEQ ID No. 1 in the sequence listing; (ii) the amino acid sequence shown in SEQ ID No.1 in the sequence listing is substituted and / or deleted and / or added with one, several or dozens of amino acid residues, and the derived protein has the same function as (i); (iii) a derivative protein having more than 90% homology with the amino acid sequence shown in SEQ ID NO.1 and having the same function as (i).

5. The method according to claim 4, characterized in that The gene of the P450 monooxygenase with the amino acid sequence shown in SEQ ID NO.1 is introduced into the microalgae by genetic engineering method, and the gene is overexpressed in the microalgae; preferably, the nucleotide sequence of the gene is shown in SEQ ID NO.

2.

6. A method for constructing microalgae with improved fucoxanthin synthesis and / or light stress response ability, characterized in that: The method comprises the step of over-expressing a P450 monooxygenase having an amino acid sequence as shown in SEQ ID NO. 1 in the microalgae.

7. The method according to claim 7, characterized in that: The nucleotide sequence of the P450 monooxygenase gene is shown in SEQ ID NO.2; the nucleotide sequence shown in SEQ ID NO.2 is connected to an overexpression vector and then transformed into microalgae.

8. The method according to claim 8, characterized in that The microalgae is Phaeochaete tricornutum; the overexpression vector is a microalgae expression vector or a plant expression vector, and a constitutive or inducible promoter drives the expression of the P450 monooxygenase gene in the microalgae.

9. A microalgae with improved fucoxanthin synthesis and / or light stress response ability, characterized in that: The method is constructed by any one of claims 4 to 6.

10. Use of the microalgae according to claim 9 in producing fucoxanthin.

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